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HEMATOPOIESIS
Cytoplasmic Domains of the Leukemia Inhibitory Factor Receptor Required
for STAT3 Activation, Differentiation, and Growth Arrest
of Myeloid Leukemic Cells
By Mikio Tomida, Toshio Heike, and Takashi Yokota
Leukemia inhibitory factor (LIF) induces growth arrest and
macrophage differentiation of mouse myeloid leukemic cells
through the functional LIF receptor (LIFR), which comprises a
heterodimeric complex of the LIFR subunit and gp130. To
identify the regions within the cytoplasmic domain of LIFR
that generate the signals for growth arrest, macrophage
differentiation, and STAT3 activation independently of gp130,
we constructed chimeric receptors by linking the transmembrane and intracellular regions of mouse LIFR to the extracellular domains of the human granulocyte macrophage colonystimulating factor receptor (hGM-CSFR) ␣ and ␤c chains.
Using the full-length cytoplasmic domain and mutants with
progressive C-terminal truncations or point mutations, we
show that the two membrane-distal tyrosines with the
YXXQ motif of LIFR are critical not only for STAT3 activation,
but also for growth arrest and differentiation of WEHI-3B Dⴙ
cells. A truncated STAT3, which acts in a dominant negative
manner was introduced into WEHI-3B Dⴙ cells expressing
GM-CSFR␣-LIFR and GM-CSFR␤c-LIFR. These cells were not
induced to differentiate by hGM-CSF. The results indicate
that STAT3 plays essential roles in the signals for growth
arrest and differentiation mediated through LIFR.
r 1999 by The American Society of Hematology.
L
Baumann et al8 constructed chimeric receptors consisting of
the extracellular domain of the granulocyte colony-stimulating
factor receptor (G-CSFR) and the cytoplasmic domain of LIFR
and showed that the cytoplasmic domain of LIFR was capable
of inducing gene expression in hepatic cells when induced to
form a homodimer. It was not known whether or not the cell
differentiation and proliferation signals were generated through
the cytoplasmic region of LIFR alone. We fused the transmembrane and intracellular regions of LIFR to the extracellular parts
of the human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) ␣ and ␤c chains, respectively. GMCSFR is composed of a specific GM-CSFR ␣ chain and a
common ␤c subunit that is shared with the receptors for IL-3
and IL-5.15 Using these chimeric receptors, we show that a
homodimer of the cytoplasmic domain of LIFR can generate the
signals for growth arrest and differentiation in mouse WEHI-3B
D⫹ and M1 myeloid leukemic cells.
Dimerization of the hematopoietin receptors initiates intracellular signaling by activating members of the receptor-associated
tyrosine kinase family, referred to as Jaks.16 The information is
next relayed by a family of transcription factors known as
STATs (signal transducers and activators of transcription).17
Tyrosine phosphorylation of the STAT proteins is necessary for
the formation of STAT homo- or heterodimers and subsequent
DNA binding. IL-6 type cytokines preferentially activate STAT-3,
STAT-1,18 and STAT-5.19 Tyrosine residues within receptors
mediate signal transduction through the recruitment of molecules containing either Src homology 2 domains or phosphotyrosine binding domains.18 There are six tyrosine residues within
the LIFR cytoplasmic domain.20,21 In this study, mutational analysis
of LIFR showed the tight correlation between STAT3 activation and
induction of differentiation of WEHI-3B D⫹ cells. Overexpression
of dominant negative STAT3 blocks the induction of growth
arrest and differentiation mediated through LIFR.
EUKEMIA INHIBITORY factor (LIF)/differentiationstimulating factor (D-factor) is a multifunctional cytokine
that was initially identified as a factor that inhibits the proliferation and induces macrophage differentiation of the murine
myeloid leukemic cell line, M1.1-4 WEHI-3B D⫹ leukemic cells
transfected with LIF receptor (LIFR) cDNA are also induced to
differentiate by LIF.5 LIF maintains embryonic stem cells in an
undifferentiated, pluripotent state, enhances the synthesis of
acute phase proteins by hepatocytes, regulates nerve differentiation, and induces cardiac myocyte hypertrophy.6-11 It is a
member of the interleukin (IL)-6 type cytokine family. Receptors for these cytokines are composed of multisubunit complexes that share a common signaling subunit, gp130. IL-6 and
IL-11 induce the homodimerization of gp130, while LIF,
cardiotrophin (CT)-1, ciliary neurotrophic factor (CNTF), and
human oncostatinM(OSM) induce gp130 heterodimer formation with the LIFR subunit.12 The essential role of gp130 in the
signaling by these receptors is demonstrated by the fact that
antibodies to gp130 are capable of neutralizing the cell responses to all of these cytokines.12 Targeted disruption of the
LIFR gene causes placental, skeletal, neural, and metabolic
defects, and thereby results in perinatal death.13,14 However, the
functional significance of the cytoplasmic domain of LIFR is
not well understood.
From the Saitama Cancer Center Research Institure, Ina, Saitama,
Japan; and the Department of Stem Cell Regulation, Institute of
Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo,
Japan.
Submitted July 8, 1998; accepted November 5, 1998.
Supported in part by a Grant-in-Aid for Scientific Research on
Priority Areas from the Ministry of Education, Science, Sports and
Culture of Japan.
Address reprint requests to Mikio Tomida, PhD, Saitama Cancer
Center Research Institute, Ina, Saitama 362-0806, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1999 by The American Society of Hematology.
0006-4971/99/9306-0005$3.00/0
1934
MATERIALS AND METHODS
Cells and cell culture. WEHI-3B D⫹ leukemic cells (kindly
provided by Dr Alan C. Sartorelli, Yale University School of Medicine,
New Haven, CT)) were cultured in McCoy’s 5A modified medium
(GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal calf
serum (FCS). M1 cells were cultured as described previously.1,2
Blood, Vol 93, No 6 (March 15), 1999: pp 1934-1941
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ROLE OF STAT3 IN DIFFERENTIATION OF WEHI-3B
Cytokines. Recombinant human GM-CSF was purchased from
Peprotech, London, England. Recombinant human IL-6 was kindly
provided by Ajinomoto Co (Kawasaki, Japan). Recombinant human
D-factor (LIF) was produced in Chinese hamster ovary cells and
purified to homogeneity as described previously.4
Plasmid construction. For the construction of pCAG-hGM-CSFR
␣-mLIFR and pMKIT-hGM-CSFR ␤c-mLIFR, the cDNA of mouse
LIFR was cloned from ES cell line A3.1 by reverse transcriptionpolymerase chain reaction (RT-PCR).21,39 The PCR products were
inserted into pSP72 (Promega, Madison, WI) at the Pvu II site and the
nucleotide sequence was confirmed using a 373A DNA sequencing
system (Perkin Elmer, Foster City, CA). The hGM-CSFR␣ (pCEV4hGMR␣) and hGM-CSFR␤c (pME18S-KH97) cDNAs were kindly
provided by Dr A. Miyajima (University of Tokyo, Tokyo, Japan). All of
these cDNAs were subcloned into pCAG22 at the Xho I site, which was
provided by Dr J. Miyazaki (Osaka University, Osaka, Japan). The
chimeric receptor constructions, which have the extracellular domain of
hGM-CSFR␣ or ␤c linked to the transmembrane and cytoplasmic
regions of mLIFR, were generated by overlap extension using PCR.23 In
brief, complementary oligonucleotide primers and PCR were used to
generate two DNA fragments with overlapping ends. In the case of
hGM-CSFR␣-mLIFR, the following two components were first generated by PCR; the extracellular domain fragment of hGM-CSFR␣ with
the first 9 bp of the transmembrane domain of mLIFR and the
transmembrane/intracellular domain of mLIFR with the last 9 bp of the
extracellular domain of hGM-CSFR␣. These fragments were combined
in a subsequent fusion reaction, in which the overlapping ends were
annealed, allowing the 38 overlap of each strand to serve as a primer for
the 38 extension of the complementary strand. The resulting fusion
product was further amplified by PCR. This PCR product was inserted
into pSP72 and its nucleotide sequence was confirmed using the 373A
DNA sequencing system. Subsequently, the following three fragments
were prepared: the first part of pCAG with the first part of hGMCSFR␣, the chimeric fragment from the latter part of hGM-CSFR␣ plus
the first part of the transmembrane/intracellular mLIFR, and the latter
part of transmembrane/intracellular mLIFR plus the latter part of
pCAG. These three fragments were ligated, resulting in completion of
the chimeric receptor under control of the CAG promoter. For constructing pMKIT-hGM-CSFR␤c-LIFR, the fragment of hGM-CSFR␤c-LIFR
with the CAG promoter was isolated and ligated at the Bam HI and Not I
sites of pMKIT neo, which was kindly provided by Dr K. Maruyama
(Tokyo Medical and Dental University, Tokyo, Japan).
Mutant receptors with progressive C-terminal truncations or cytoplasmic domain tyrosine = phenylalanine substitutions were constructed
(see Fig 2). Chimeric cDNAs were subcloned into the Xho I site of
pGEM-7Zf (Promega). C-terminal truncations were generated by PCR
using a 58oligonucleotide (MDR1F: 58-CTGTAAGGCGCTACAGTTTCAGAA-38) located upstream of the unique Bgl II site in
LIFR cDNA, and 38 oligonucleotides introducing termination codons
and Bst EII sites (T112: 58-ATGGTGACCTACTGCACATCGATGTACACC-38; T129: 58-ATGGTGACCTACTCTGCTTTGGCTTGCGGC38; and T149: 58-ATGGTGACCTAGGGAAGGCGCATCTGTGG-38).
Receptor fragments with tyrosine = phenylalanine substitutions were
generated by recombinant PCR24 using MDR1F and MDR5R (58GACAAAGGGTGACCTGGTTA-38, which includes the normal termination codon and a Bst EII site) as external primers. The following
oligonucleotides were used as internal primers: F5F, 58-GTGGCAGGCTTTAAGCCACAG-38; F5R, 58-CTGTGGCTTAAAGCCTGCCAC-38; F4F,
58-CAGTCCATGTTTCAGCCGCAA-38; F4R, 58-TTGCGGCTGAAACATGGACTG-38; F3F, 58-CAGGTGGTGTTCATCGATGTG-38; F3R, 58CACATCGATGAACACCACCTG-38; F6F, 58-ACCGCCGGTTTCAGACCTCAG-38; and F6R, 58-CTGAGGTCTGAAACCGGCGGT-38.
Construct F3/4/5/6 was generated by recombination between F3
plasmid DNA and a fragment of F4/5/6 digested with Cla I and Bst EII.
Similarly, F4/5/6 was generated by recombination between F4 and F5/6
1935
at the Bam HI and Bst EII sites. F5/6 was generated by PCR using F5
plasmid DNA as a template, and F6F and F6R as primers. The
nucleotide sequences of the fragments derived on the PCR were
confirmed by dideoxy sequencing using an ALF DNA sequencer
(Pharmacia, Uppsala, Sweden). The construct of dominant negative
STAT325 was kindly provided by Dr Alice L.-F.Mui (DNAX Research
Institute, Palo Alto, CA).
Transfection of DNA. DNA was introduced into cells by electroporation using a Gene Pulser (Bio-Rad, Richmond, CA). The cells (107)
were suspended in 0.8 mL of HEPES-buffered saline (50 mmol/L
HEPES, 137 mmol/L NaCl, 6.8 mmol/L KCl, 0.28 mmol/L Na2 HPO4,
and 0.1% dextrose, pH 7.1) containing 25 µg each of the expression
plasmids for chimeric GM-CSFR ␣ and ␤c. The cells were exposed to a
450 V (WEHI-3B D⫹) or 400 V (M1) pulse with a capacitance of 960
µF. After 2 days culture, the cells were transferred to 96-well plates and
then cultured in medium containing G-418 at the final concentration of
1.8 mg/mL. Surface expression of the transfected chimeric receptor genes
were analyzed by flow cytometry with an Epics XL (Coulter Electronics,
Luton, UK), using anti–GM-CSFR␣ (S-20; Santa Cruz Biotechnology, Santa
Cruz, CA) and anti–GM-CSFR ␤c (S-16; Santa Cruz Biotechnology).
Transfectants expressing W238 receptors were further transfected
with 30 µg of pME18S ⌬STAT3B together with 1 µg of pPUR
(Clontech, Palo Alto, CA), which carries the puromycin-resistant gene.
The cells were selected in a medium containing 2 µg/mL puromycin and
1.8 mg/mL G418. The C-terminal truncated STAT3 protein was immunoprecipitated using anti-STAT3 antibody K15 (directed against amino acids
626-640), which was obtained from Santa Cruz Biotechnology.
Properties of differentiated cells. The differentiation of M1 cells
was assayed by measuring the induction of phagocytic activity in the
cells. M1 cells (3 ⫻ 105 cells/mL) were incubated with cytokines for 2
days. The cells were harvested, suspended in serum-free medium
containing 0.2% of a suspension of polystyrene latex particles (average
diameter, 0.944 µm, Seradyn, Indianapolis, IN), and then incubated for
4 hours at 37°C. The cells were then thoroughly washed three times
with phosphate-buffered saline (PBS). Cells containing more than 10
latex particles were scored as phagocytic cells.
The ability of cells to reduce nitroblue tetrazolium (NBT) was used as
a functional marker of the differentiation of WEHI-3B D⫹ cells. The
cells (104 cells/mL) were incubated with cytokines for 4 days. The cells
were harvested, and the cell numbers were determined using a Model
ZM Coulter Counter. The cells were suspended in 1 mL of serum-free
medium containing 1 mg/mL of NBT and 100 ng/mL of 12-O-tetra
decanoylphorbol 13-acetate, and then incubated for 1 hour at 37°C. The
reaction was stopped by adding HCl to the final concentration of
1 mol/L. The suspension was centrifuged, and the formazan deposits
were solubilized by adding dimethyl sulfoxide. The absorption of the
formazan solution at 560 nm was measured with a spectrophotometer.
Agar cultures of WEHI-3B D⫹ cells were performed in 35-mm Petri
dishes, as described previously.26 Briefly, 200 cells were cultured in
1 mL of 0.3% agar (DIFCO Laboratories, Detroit, MI) in Dulbecco’s
modified Eagle’s medium containing 20% FCS and various concentrations of hGM-CSF or IL-6 (100 ng/mL) as a positive inducer of
differentiation. Colony numbers and morphology were scored after
7 days incubation at 37°C under a fully humidified atmosphere of
5% CO2 in air. Aggregates of more than 50 cells were scored as
colonies. Colonies with a halo of dispersed cells were scored as
differentiated.
Immunoprecipitation and Western blot analysis. WEHI-3B D⫹
cells (107) were incubated with or without hGM-CSF (100 ng/mL) for
10 minutes at 37°C. The reaction was stopped by adding ice-cold PBS.
The cells were harvested, washed once with ice-cold PBS, and then
lysed in 400 µL of lysis buffer (1% Triton X-100, 150 mmol/L NaCl, 20
mmol/L Tris-HCl, pH 7.4, 2 mmol/L EDTA, 1 mmol/L Na3VO4,
1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 10
µg/mL pepstatin, 1 µg/mL aprotinin, and 100 µg/mL Pefabloc) for 5
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1936
TOMIDA, HEIKE, AND YOKOTA
Table 1. Differentiation of M1 Cells Expressing GM-CSFR␣-LIFR
and GM-CSFR␤-LIFR
Cytokine
(ng/mL)
Phagocytic Cells (%)
None
GM-CSF
GM-CSF
GM-CSF
LIF
IL-6
2.5
5
10
2
10
1.3 ⫾ 0.7*
27.5 ⫾ 9.0
36.8 ⫾ 4.3
48.0 ⫾ 6.0
22.8 ⫾ 4.8
39.4 ⫾ 7.6
Cells were incubated with cytokines for 2 days. Phagocytic activity
of the cells was assayed as described in Materials and Methods.
*Each value is the mean of duplicate determinations ⫾ standard
error.
RESULTS
Signal transduction through the chimeric receptor carrying
the full-length cytoplasmic domain of LIFR in M1 and WEHI-3B
D⫹ cells. Stimulation of M1 cells with LIF or IL-6 induces
macrophage differentiation and growth arrest.1,2,27-29 Although
wild-type WEHI-3B D⫹ cells respond to IL-6, but not LIF, cells
transfected with LIFR cDNA respond to both LIF and IL-6.5 M1
and WEHI-3B D⫹ cells were transfected with both the hGMCSFR␣-LIFR and hGM-CSFR␤c-LIFR constructs, and expression of these receptors was determined by flow cytometric
analysis (Fig 1). The differentiation of M1 cells was assessed by
measuring the induction of phagocytic activity in the cells, as
the induction of phagocytic activity in the cells was associated
with the induction of other phenotypic markers of cell differentiation.1 M1 cells expressing both the ␣ and ␤c chimeric
receptors responded to hGM-CSF, as well as LIF and IL-6
(Table 1). The differentiation of WEHI-3B D⫹ cells was
Fig 1. Flow cytometric analysis of chimeric receptor expression in
M1 transformants. M1 cells were transfected with GM-CSFR␣-LIFR
and GM-CSFR ␤c-LIFR cDNA. The cells were incubated with anti–GMCSFR ␣ (B) or anti–GM-CSFR ␤c (C). Antibody binding was detected
by incubation with fluorescein-conjugated rabbit antimouse immunoglobulin. Samples were analyzed using a flow cytometer. (A) The flow
cytometry profile stained with the fluorescein-conjugated second antibody alone.
minutes on ice. Cell lysates were centrifuged for 5 minutes at 4°C at
14,000 rpm, and the resulting supernatants were incubated with
antibodies to STAT3 (C-20), STAT5b (C-17), SHP-2 (Santa Cruz
Biotechnology), or Shc (Transduction Laboratories, Lexington, KY) for
2 hours at 4°C. Protein A-Sepharose was added to the reaction mixture,
and the incubation was continued overnight at 4°C. The beads were
pelleted by centrifugation and then washed three times with cold PBS. The
immune complexes were eluted with 20 µL of Laemmli sample buffer,
resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and then transferred to Immobilon P (Millipore, Bedford,
MA). The membrane was incubated with antiphosphotyrosine antibodies
(4G10; Upstate Biotechnology, Lake Placid, NY) for 1 hour. After washing,
the membrane was incubated with horseradish peroxidase-conjugated secondantibodies for 1 hour. The immune complex was detected using an enhanced
chemiluminescence system (ECL; Amersham Life Science, Little Chalfont,
UK). Blots were stripped with 2% SDS, 100 mmol/L 2-mercaptothanol, and
62.5 mmol/L Tris-HCl, reblocked, and then reprobed with antibodies to
STAT3 (Transduction Laboratories).
Fig 2. Deletion and point mutations in LIFR. The cytoplasmic
regions of the LIFR mutants are schematically shown. The transmembrane domain (TM), putative box 1 (B1), box 2 (B2), and box 3 (B3),
and the positions of tyrosine (Y) and phenylalanine (F) residues are
indicated.
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ROLE OF STAT3 IN DIFFERENTIATION OF WEHI-3B
1937
Fig 3. Differentiation of
WEHI-3B Dⴙ cells expressing LIFR
mutants. Cells expressing various chimeric receptors were
treated for 4 days with 10 ng/mL
of hGM-CSF or 100 ng/mL of
IL-6. The NBT-reducing activity
of the cells was determined by
the colorimetric assay. The data
for representative clones are presented as percentages of the values for untreated control cultures. The values represent the
averages of duplicate assays ⴞ
standard error.
assessed first by measuring the ability of the cells to reduce
NBT, a differentiation marker. The cells expressing both the ␣
and ␤c chimeric receptors exhibited enhanced ability to reduce
NBT on treatment with hGM-CSF, as well as IL-6 (see W238 in
Fig 3). Cell migration and growth arrest of the transfected cells
were also induced by treatment with hGM-CSF in a soft agar
culture (see W238 in Fig 4). These results suggest that
homodimerization of the LIFR cytoplasmic domain is sufficient
to induce macrophage differentiation of M1 and WEHI-3B D⫹
cells because hGM-CSF cannot bind to mouse GM-CSFR.
Cytoplasmic domains that are required for inducing differentiation and growth arrest of WEHI-3B D⫹ cells. To determine
the receptor domains required for growth arrest and differentiation, LIFR mutants, as shown in Fig 2, were constructed. ␣ and ␤c
chimeric receptor cDNAs carrying these LIFR mutations were
transfected into WEHI-3B D⫹ cells. hGM-CSF could induce
differentiation and growth arrest of the transformants expressing
truncated forms of the LIFR cytoplasmic domain containing at least
149 amino acid residues (Figs 3 and 4). More extensively truncated
forms of the LIFR (T129 and T119) were unable to generate signals
for differentiation and cell growth arrest. A normal differentiation
response to IL-6 was observed in WEHI-3B D⫹ cells expressing
each of the various mutant receptors.
The phosphorylation of tyrosine residues is considered to be
a crucial process in the initiation of signal transduction from the
receptors after ligand binding. LIFR has six tyrosine residues in
its cytoplasmic domain (Fig 2). The third tyrosine residue from
the membrane-proximal region is located in a YXXV motif, a
known docking site for SHP-2. Three tyrosine residues, ie, the
fourth, fifth, and sixth ones, are located in a YXXQ motif, a STAT3
consensus binding sequence.18 We introduced a mutation into either
the third (F3), fourth (F4), or fifth (F5) tyrosine residue, or into four
tyrosine residues, ie, the third, fourth, fifth, and sixth ones (F3/4/5/6).
Mutants, F3, F4, and F5, but not F3/4/5/6, could induce growth
arrest and macrophage differentiation (Figs 3 and 4).
STAT3 activation in relation to growth arrest and differentiation. LIF and related cytokines activate a common set of
Fig 4. Soft agar colony assaying of WEHI-3B Dⴙ cells expressing LIFR mutants. Cells expressing the various chimeric receptors (W238, T149, T129, T119, F3, F4, F5, or F3/4/5/6) were cultured in agar with increasing concentrations of hGM-CSF or IL-6
(100 ng/mL). The colony numbers (left panels) and the proportions of colonies containing differentiated cells (right panels)
were determined after 7 days. The means of duplicate assays are
shown.
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1938
TOMIDA, HEIKE, AND YOKOTA
Fig 5. Tyrosine phosphorylation of STAT3 through the chimeric receptors. Cells expressing various chimeric receptors were either stimulated
(ⴙ) with hGM-CSF (10 ng/mL) for 10 minutes or left unstimulated (-). STAT3 was immunoprecipitated from cell lysates using anti-STAT3
antibodies and then probed with either antiphosphotyrosine antibodies (upper panel) or anti-STAT3 antibodies (lower panel). The immune
complex was visualized by chemiluminescence.
signalling molecules including components of the JAK-STAT
pathway and of the Ras-MAP kinase signalling cascade.9,11,12 To
assess the ability of the receptor mutants to activate Shc and
SHP-2 in the Ras-MAP kinase pathway, and STAT3 and STAT5,
Western blot analysis was performed on lysates of stimulated
and unstimulated WEHI-3B D⫹ cells. Shc, SHP-2, and STAT5
were already phosphorylated in all cell lines cultured in medium
containing FCS. Stimulation with hGM-CSF did not cause an
increase in the level of tyrosine phosphorylation of these
proteins (data not shown). On the contrary, phosphorylation of
STAT3 was not detected in unstimulated cells. Stimulation with
hGM-CSF induced the tyrosine phosphorylation of STAT3 in
the transformants of W238 , T149, F3, F4, and F5, but not in the
transformants of T129, T119, and F3/4/5/6 (Fig 5). The results
show that the tight correlation between STAT3 activation and
induction of differentiation and growth arrest of the cells.
Effect of dominant-negative STAT3 on growth arrest and
differentiation. To examine the role of STAT3, a truncated
STAT3 (⌬STAT3), which lacks the transactivation domain and
acts as a dominant negative manner was generated.25 The
construct was transfected with a puromycin-resistance selection
marker into WEHI-3B D⫹ cells expressing W238 receptors.
Transfectants that were resistant to both puromycin and neomycin were isolated. The responsiveness of the cells to IL-6 was
first examined because dominant negative forms of STAT3 were
shown to block the IL-6–mediated growth arrest and differentiation of M1 cells.28,29 Several clones exhibiting different sensitivities to IL-6 were selected, and the expression of truncated
A
STAT3 in these clones was examined by immunoblot analysis.
The expression of truncated STAT3 correlated with a decrease
in sensitivity to IL-6. To examine the activation of STAT3, cells
were stimulated with IL-6 or hGM-CSF. In the cells overexpressing
truncated STAT3 (Fig 6B), the tyrosine-phosphorylation of
endogenous STAT3 in response to IL-6 was reduced and the
response to hGM-CSF was almost completely blocked (Fig
6A). We examined the effect of expression of the mutant STAT3
protein on the induction of differentiation and growth arrest of
WEHI-3B D⫹ cells. As shown in Figs 7 and 8, in the cells
expressing dominant negative STAT3, the induction of differentiation and growth arrest by hGM-CSF or IL-6 was dramatically
reduced relative to the control. These results suggest that STAT3
plays an essential role in the signals mediated by the LIFR
homodimer.
DISCUSSION
LIFR is a member of the hematopoietin receptor family.20 It is
structurally most similar to gp130, G-CSFR, IL-12 receptor,
OSM receptor, and leptin receptor.12,30,31 Among these receptors, gp130 and G-CSFR have been extensively studied as to the
cytoplasmic domains required for cell proliferation and differentiation.27-29,32,33 While these receptor subunits function as homodimers, LIFR functions as a heterodimer with gp130. To
examine signal transduction by LIF, we fused the cytoplasmic
domain of mouse LIFR or gp130 to the extracellular domain of
the human GM-CSFR␣ and ␤c chains, respectively. By transfection of different combinations of chimeric receptors into mouse
B
Fig 6. Inhibition of tyrosine phosphorylation of STAT3 by dominant negative STAT3. (A) WEHI-3B Dⴙ cells expressing the chimeric LIFR (W238)
and ⌬STAT3 or the chimeric LIFR alone (PurrNeor) were stimulated with hGM-CSF (10 ng/mL) or IL-6 (100 ng/mL) for 10 minutes. STAT3 was
immunoprecipitated from cell lysates using anti-STAT3 antibodies (C20; Santa Cruz Biotechnology), and then probed with either antiphosphotyrosine antibodies or anti-STAT3 antibodies (Transduction Laboratories). (B) Expression of ⌬STAT3 in WEHI-3B Dⴙ cells. STAT3 was
immunoprecipitated from cell lysates using anti-STAT3 antibodies (K-15; Santa Cruz Biotechnology) and then probed with anti-STAT3 antibodies
(Transduction Laboratories).
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ROLE OF STAT3 IN DIFFERENTIATION OF WEHI-3B
Fig 7. Inhibition of differentiation of WEHI-3B Dⴙ cells expressing
LIFR by dominant negative STAT3. Cells expressing LIFR (W238) and
⌬STAT3, or LIFR alone (PurrNeor) were treated for 4 days with 10
ng/mL of hGM-CSF or 100 ng/mL of IL-6. The NBT-reducing activity of
the cells was determined by the colorimetric assay. The data for
representative clones are presented as percentages of the values for
untreated control cultures. The values represent the averages of
duplicate assays ⴞ standard error.
cells, we analyzed the signals of not only homodimers, but also
heterodimers of LIFR and gp130.39
In the present study, we transfected M1 and WEHI-3B D⫹
cells with GM-CSFR␣-LIFR and GM-CSFR␤c-LIFR cDNA
and found that hGM-CSF induced macrophage differentiation
and growth arrest of these leukemic cells, suggesting that the
cytoplasmic domain of LIFR is capable of signal transduction
Fig 8. Soft agar colony assaying of WEHI-3B Dⴙ
cells expressing LIFR and dominant negative STAT3.
Cells expressing LIFR (W238) and ⌬STAT3, or LIFR
alone (PurrNeor) were cultured in agar with increasing concentrations of hGM-CSF or IL-6 (100 ng/mL).
The colony numbers (left panels) and the proportions
of colonies containing differentiated cells (right panels) were determined after 7 days. The means of
duplicate assays are shown.
1939
when it is induced to form a homodimer. Therefore, mutants of
these chimeric receptors permit analysis of the functional
domain of the LIFR cytoplasmic domain in the absence of
gp130. We identified the first 149 amino acid residues of the
cytoplasmic domain of LIFR as the minimal region necessary
for growth arrest and differentiation of WEHI-3B D⫹ cells. The
amino acid 136-145 region, called box 3, is conserved among
gp130, LIFR and G-CSFR and contains the fifth tyrosine
residue and a YXXQ motif. The tyrosine residue in the motif
was shown to play a role in activating STAT3.18 The fourth and
sixth tyrosine residues are also contained in the YXXQ motif.21
However, the truncated mutant, T129, containing the fourth
tyrosine residue and a YXXQ motif, neither activated STAT3
nor induced the differentiation of WEHI-3B D⫹ cells. The
mutant, T149, with the F5 mutation was also inactive (data not
shown). Therefore, the sixth tyrosine residue may substitute for
the fifth tyrosine residue in activating STAT3 and the generation
of the signals for growth arrest and differentiation because the
F5 mutation in the full-length cytoplasmic domain did not affect
the function of the receptor (Figs 3 and 4).
Kuropatwinski et al19 and Stahl et al18 fused the cytoplasmic
domain of LIFR and the extracellular domains of G-CSFR and
the epidermal growth factor receptor, respectively. These chimeric receptors formed homodimers after binding to ligands.
They showed that similar constructs to our T129 mutant could
activate STAT3 in COS-1 or COS-7 cells. The difference
between their and our present results seems to originate from
the difference between the lineages of the cells used.
Our present study showed that activation of STAT3 is tightly
correlated with the signals for growth arrest and macrophage
differentiation, and that a dominant negative form of STAT3
inhibited both LIFR-mediated growth arrest and macrophage
differentiation of the WEHI-3B D⫹ transformants. These results
are consistent with the results obtained by Nakajima et al28 and
Minami et al.29 They showed that activation of STAT3 is
essential for gp130-mediated growth arrest and differentiation
of M1 cells. On the other hand, Alexander et al34 showed that
activation of STAT3 DNA binding was insufficient for c-Mpl
From www.bloodjournal.org by guest on April 18, 2017. For personal use only.
1940
TOMIDA, HEIKE, AND YOKOTA
(thrombopoietin receptor)-mediated WEHI-3B D⫹ differentiation. They suggested that activation of components of the Ras
signalling cascade, initiated by interaction of Shc with c-Mpl,
plays a decisive role in the differentiation signals. Therefore, we
examined Shc phosphorylation in our WEHI-3B D⫹ cells. In
contrast to their results, Shc was phosphorylated in the cells
cultured in normal medium containing FCS. Treatment of the
cells with hGM-CSF did not cause a further increase in
phosphorylation. Nicholson et al33 analyzed tyrosine residues in
G-CSFR that mediate the induction of differentiation of M1
cells. They showed that STAT3 was activated by G-CSF in the
cells expressing G-CSFR tyrosine mutants unable to mediate
G-CSF–induced differentiation. Therefore, activation of STAT3
is not sufficient for G-CSF–mediated M1 differentiation. Tyrosine phosphorylation of Shc was not required for the differentiation of M1 cells. All of these results, including ours, suggest that
activation of STAT3 is required, although not sufficient, for
induction of the differentiation of myeloid leukemic cells such
as WEHI-3B D⫹ and M1. Multiple signaling pathways through
LIF in various cells have been reported.35-37
Recently, Starr et al38 reported that homodimers of the LIFR
cytoplasmic domain were able to deliver the signal that blocks
differentiation of ES cells, although they were not sufficient for
transducing a differentiation signal in M1 cells nor for induction
of a proliferation signal in Ba/F3 cells. We also examined the
effects of our chimeric receptors on ES and Ba/F3 cells.39
Homodimers of the LIFR cytoplasmic domain could not
support the proliferation of these cells. The reason for the
difference between our results and theirs is not known, but
clonal variation of ES and M1 cells and differences in the
selection of transfectants may be possible explanations. In
general, LIFR homodimers may be less efficient than heterodimers of LIFR and gp130 in signaling biological responses.
However, in the case of WEHI-3B D⫹ cells, LIFR homodimers
were equivalent in activity to heterodimers of LIFR and gp130.
The response to hGM-CSF of WEHI-3BD⫹ cells transfected
with the present chimeric receptors was very similar to the
response to LIF of WEHI-3B D⫹ cells transfected with the
full-length LIFR, which formed a heteromeric complex with
endogenous gp130.5
We showed that the cytoplasmic domain of LIFR in the
absence of gp130 could generate the signals for growth arrest
and differentiation of myeloid leukemic cells. It is possible that
an as yet undiscovered cytokine could induce downstream
signaling through homodimerization of LIFR in some cell
types. No signaling pathway specific to LIFR has been identified. Our chimeric receptors will be useful for elucidating the
different signaling potentials of LIFR versus gp130.
ACKNOWLEDGMENT
We thank T. Higashihara for her technical assistance and Dr T.
Matsuda for the helpful discussions.
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1999 93: 1934-1941
Cytoplasmic Domains of the Leukemia Inhibitory Factor Receptor Required
for STAT3 Activation, Differentiation, and Growth Arrest of Myeloid
Leukemic Cells
Mikio Tomida, Toshio Heike and Takashi Yokota
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